Presynaptic calcium channels are vital elements that regulate communication at virtually all chemical synapses. Only in the last couple of years has investigation of these channels advanced to the molecular level. In this context, an important contribution has been the recent cloning by this group of a novel protein, CCCS1, that is essential for the normal operation of presynaptic calcium channels in Torpedo (fish). CCCS1 is either an integral subunit or regulator of these channels. Interestingly, database searches revealed that CCCS1 homologs of undetermined function had previously been cloned in Drosophila. These proteins, call cysteine-string proteins (csps), had been identified using monoclonal antibodies and were concentrated at Drosophila nerve terminal membranes. Preliminary results (obtained in collaboration with the discoverers of the Csp gene) indicate that deletions at the csp locus lead to defects in neuromuscular transmission that can be traced to a dysfunction of nerve ending calcium channels. The goal of this proposal is to confirm and extend these dramatic results. First CCCS1 and the csps will be co-expressed with other calcium channel subunits in frog oocytes or cultured mammalian cells. Electrophysiological characteristics of the calcium currents of these cells will give insights into the molecular interactions of these proteins. Second, a comprehensive investigation will be made of synaptic transmission in Drosophila csp mutants versus rescued and wild type controls. This will refine our understanding of the cellular consequences attending presynaptic defects in calcium channels and the csps. Third, calcium channels of the cell bodies of neurons from mutant and control flies will be studied by patch clamp to determine if there are alterations of channel properties that may help to understand csp function. Taken together, these experiments lay the foundation for a wide range of new approaches to study presynaptic activity. Moreover, it can hardly escape attention that the severely afflicted csp mutants (which barely survive to adulthood even when reared at 16-18 degrees C) may be useful models for congenital neurological diseases. And, ultimately one can hope that investigations of this new family of cysteine-string proteins will have beneficial ramifications for learning and memory disorders, mental retardation, myasthenic syndromes and other disorders of the synapse.